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Reversible Protonated Resting State of the Nitrogenase Active Site Christine N. Morrison,† Thomas Spatzal,†,‡ and Douglas C. Rees*,†,‡ †
Division of Chemistry and Chemical Engineering and ‡Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, United States S Supporting Information *
ABSTRACT: Protonated states of the nitrogenase active site are mechanistically significant since substrate reduction is invariably accompanied by proton uptake. We report the low pH characterization by X-ray crystallography and EPR spectroscopy of the nitrogenase molybdenum iron (MoFe) proteins from two phylogenetically distinct nitrogenases (Azotobacter vinelandii, Av, and Clostridium pasteurianum, Cp) at pHs between 4.5 and 8. X-ray data at pHs of 4.5−6 reveal the repositioning of side chains along one side of the FeMo-cofactor, and the corresponding EPR data shows a new S = 3/2 spin system with spectral features similar to a state previously observed during catalytic turnover. The structural changes suggest that FeMo-cofactor belt sulfurs S3A or S5A are potential protonation sites. Notably, the observed structural and electronic low pH changes are correlated and reversible. The detailed structural rearrangements differ between the two MoFe proteins, which may reflect differences in potential protonation sites at the active site among nitrogenase species. These observations emphasize the benefits of investigating multiple nitrogenase species. Our experimental data suggest that reversible protonation of the resting state is likely occurring, and we term this state “E0H+”, following the Lowe−Thorneley naming scheme.
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INTRODUCTION Nitrogen fixation is the process of breaking the kinetically inert N−N triple bond via either reduction or oxidation of dinitrogen. Biologically, nitrogen fixation is accomplished by the enzyme nitrogenase to yield ammonia, with an overall reaction stoichiometry conventionally described by eq 1:
Electron paramagnetic resonance (EPR) is a powerful tool for studying the electronic states of the FeMo-cofactor since the E0 state exhibits a strong, unique rhombic spectrum, resulting from transitions within the ±1/2 ground-state Kramers’ doublet of a S = 3/2 system.7 In contrast, the P-cluster is diamagnetic in the dithionite-reduced form (PN) and exhibits a weak resonance at g = 12 in the oxidized form (Pox).8,9 The reported EPR spectra of the FeMo-cofactor under turnover conditions include three spin systems called 1a, 1b, and 1c.10−12 1a is the resting state (E0), and 1b and 1c, which are in equilibrium with 1a,12 are attributed to E2 and are thought to represent different states of the FeMo-cofactor during turnover. More specifically, 1c has been suggested to result from protonation of the FeMo-cofactor.11 The E1 state is EPR-silent. The FeMo-cofactor (Figure 1) exhibits approximate C3v symmetry, with the core provided by a trigonal prism of six Fe atoms (Fe2−7) surrounding an interstitial carbon.13−15 Each face of the trigonal prism is bridged by one of three “belt” S labeled S2B, S3A and S5A. Crystallographic evidence for turnover-dependent rearrangements of belt sulfurs is demonstrated by the reversible displacement of S2B upon CO inhibition.16 Se from selenocyanate may also substitute S2B.17 In the presence of substrate and under turnover conditions, interchange of the belt sulfurs was established such that Se originally at S2B migrates to S5A and S3A before ultimately exiting the FeMo-cofactor.17 Intriguingly, the S2B site displaced
N2 + 10H+ + 8e− + 16ATP → 2NH4 + + H 2 + 16ADP + 16Pi
(1)
Nitrogenase is a highly oxygen-sensitive enzyme present in specialized microorganisms; it consists of two proteins called the molybdenum−iron (MoFe) and iron (Fe) proteins.1−3 The Fe protein contains two nucleotide binding sites and a 4Fe:4S cluster. The MoFe protein incorporates two 8Fe:7S “P-clusters” and two 7Fe:9S:C:Mo:R-homocitrate “FeMo-cofactors”, the latter of which represents the active site where substrates bind and are reduced. ATP-dependent electron transfer occurs from the 4Fe:4S cluster to the P-cluster during docking interactions between the Fe and MoFe proteins, after which the proteins separate.4−6 Substrates can only bind to forms of the FeMocofactor more reduced than the resting state. These states are conventionally designated as En, where n represents the number of electrons transferred to the MoFe protein (per active site), and E0 is the resting state.5 Following the Lowe−Thorneley model, dinitrogen binds to the FeMo-cofactor in the E3 and E4 states; however, other substrates, such as acetylene, may bind to the FeMo-cofactor in less highly reduced states.5 © 2017 American Chemical Society
Received: June 1, 2017 Published: July 10, 2017 10856
DOI: 10.1021/jacs.7b05695 J. Am. Chem. Soc. 2017, 139, 10856−10862
Article
Journal of the American Chemical Society
structure and EPR spectra of the MoFe protein have not to our knowledge been detailed, likely as it has been reported that the MoFe protein is inactivated below pH 6.2.19 However, our study shows that impacts to the atomic and electronic structure are reversible between pH 4.5 and pH 8 under the tested experimental conditions. In this study, we examine the two phylogenetically distinct nitrogenase MoFe proteins from Azotobacter vinelandii (Av1) and Clostridium pasteurianum (Cp1), which have a sequence identity of ∼36%.20 Working with Cp1 and Av1, we combine a structural approach with EPR spectroscopy to examine the atomic and electronic structure of MoFe proteins at pH 5, where the proton concentration is 2−3 orders of magnitude greater than that of typical enzyme activity measurements. Changes occurring in the MoFe protein at low pH might therefore provide crucial information about the atomic and electronic structure of the protein at an early stage of substrate reduction.
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RESULTS AND DISCUSSION Over the pH range between 4.5 and 5.8, X-ray crystal structures of Cp1 and Av1 (Table 1) reveal structural rearrangements near the Fe3,4,5,7 face of the FeMo-cofactor (Figure 2) that are fully reversible upon returning to pH ∼ 8. For these studies, the purified protein was resuspended in a low pH tribuffer system,21 allowing the pH of the protein solution to be varied from pH 2 to pH 7 with minimal variation in the ionic strength and buffer components. Av1 and Cp1 exhibit a partially and fully occupied low pH conformer, respectively, when pH ≤ 5. We determined the pH 5 structures of Cp1 and Av1 at resolutions of 1.85 and 2.30 Å, PDB IDs 5VPW and 5VQ4, respectively. At pH ∼ 6.5, Cp1 exhibits both conformations; the PDB ID for this structure is 5VQ3. The conversion of the pH ∼ 8 conformer to the low pH conformer under different pH and ionic strength conditions was explored in Cp1 over a large number of conditions. It was found that higher ionic strength contributes to increased occupancy of the low pH conformer, which occurred at pH 5.8 or lower, depending on ionic strength. In view of the dependence of conformer occupancy on pH and ionic strength as well as the challenges of measuring pH in small volumes
Figure 1. Structure of the FeMo-cofactor. The atoms of the cluster are shown in spheres and colored by element (Fe, orange; S, yellow; C, gray; Mo, cyan). Fe sites in the trigonal prism around the interstitial carbon are labeled with bold print. Belt S are also labeled and underlined. Coordinating residues and the R-homocitrate are shown in sticks and colored by element (C, gray; O, red; N, blue).
by CO bridges Fe2 and Fe6, which have been shown to be more oxidized in the resting state,18 suggesting that their reduction is critical for ligand binding at this site. There is still a high level of uncertainty in the mechanistic description of biological nitrogen fixation, including possible structural rearrangements in the FeMo-cofactor. The challenge has been to generate significant populations of higher En states competent for substrate binding. As formation of these states is associated with proton uptake, we reasoned that by studying the MoFe protein at low pH (high proton concentration), features of the active site that are characteristic of more highly reduced forms might be stabilized through Le Chatelier’s principle. The effects of low pH (pH ≤ 5) on the X-ray
Table 1. X-ray Crystallographic Data Collection and Refinement Statistics
a
Av1 at pH 5 (5VQ4)
Cp1 at pH 5 (5VPW)
Cp1 at pH 6.5 (5VQ3)
Data Collection space group cell dimensions a, b, c (Å); α, β, γ (deg) resolution (Å) Rmerge I/σ(I) completeness (%) no. unique reflections redundancy
P21 81.31, 128.9, 108.4 90, 110.9, 90 39.54−2.30 (2.30−2.34)a 0.174 (0.720)a 9.2 (3.1)a 98.8 (99.4)a 91,309 (4,321)a 6.7 (7.1)a
P21 69.62, 146.3, 116.7 90, 103.6, 90 39.20−1.85 (1.88−1.85)a 0.105 (0.684)a 11.6 (2.5)a 98.4 (95.4)a 189,858 (1,197)a 6.5 (6.2)a
P21 69.48, 148.0, 116.7 90, 103.5, 90 39.83−1.75 (1.75−1.72)a 0.079 (0.682)a 13.5 (2.9)a 98.4 (98.4)a 238,230 (11,876)a 6.8 (7.0)a
Refinement Rwork/Rfree average B-factor rms bond lengths (Å) rms bond angles (deg)
0.176/0.226 24.0 0.011 1.39
0.167/0.201 30.0 0.012 1.41
0.159/0.185 29.0 0.013 1.52
Highest resolution shell is shown in parentheses. 10857
DOI: 10.1021/jacs.7b05695 J. Am. Chem. Soc. 2017, 139, 10856−10862
Article
Journal of the American Chemical Society
Figure 2. (a) Overview of the structural rearrangements observed at low pH at the active sites of Cp1 and Av1. Both changes occur on the Fe3,4,5,7 face of the FeMo-cofactor, which is the same face that is exposed to water molecules and connects to the interstitial water channel illustrated for Cp1 (dashed black line). (b) In Cp1, a peptide flip occurs between α-Arg347 and α-Ser346, and the Arg side chain relinquishes its hydrogen bond with S5A. (c) In Av1, the α-His274 side chain swings closer to the FeMo-cofactor and displaces a water molecule; two water molecules fill the former αHis274 side chain position. The α-His274 coordinates to S5A of the FeMo-cofactor through a hydrogen-bond bridge with a water molecule. In all images, transparent gray represents the physiological pH structures. Nontransparent gray sticks show the low pH structural changes. The FeMocofactor and pH-affected residues are displayed as sticks and colored by element (yellow, S; orange, Fe;, cyan, Mo; gray, C). Water molecules are represented as red spheres. The blue meshes in (b) and (c) show the electron density maps of the pH-affected residues contoured to 2.0 and 1.5 σ, respectively.
Arg347 and S3A and S5A in Cp1 (Figure 3): S5A loses its only hydrogen bond to NH1; S3A loses its contact with the backbone amide NH; and S3A gains contacts with NH1 and NE of the arginine side chain. In Av1 at low pH, the side chain of Av1 α-His274 (adjacent to the FeMo-cofactor ligand αCys275 and corresponding to Cp1 α-Gln261) moves closer to the FeMo-cofactor and displaces a water molecule. At this new position, a water molecule bridges the Av1 α-His274 side chain and S5A of the FeMo-cofactor (Figure 2c). Of the two residues most affected by low pH in Cp1 and Av1, Cp1 α-Arg347 is invariant in all nitrogenases, whereas Av1 α-His274 is variant and exists as a glutamine residue in Cp1.20 Mutagenesis of these residues in Av1 significantly reduces substrate reduction,23,24 and α-His274 has been implicated in FeMo-cofactor insertion during Av1 assembly.25 The low pH structural rearrangements only occur on the face of the FeMo-cofactor that is exposed to water molecules (Fe3,4,5,7), potentially implicating this water pool (and likely the water channel that connects this pool to the protein surface) in proton transport between the active site and the exterior.26−28 Additionally, there is slight movement (
